Atherosclerosis, the process of vascular wall thickening and hardening, is a significant contributing factor in the development of cardiovascular disease (the leading cause of morbidity and mortality in U.S.). Therefore, understanding the molecular mechanisms involved in atherosclerotic lesion (atheroma) progression has significant relevance in human disease and therapeutic development. We recently identified a novel role for the endocytic adaptor protein, epsin, as a pro-atherogenic and pro-inflammatory regulator in the endothelium. Specifically, we observed that epsins 1 and 2 are upregulated in the aortic atheroma of western diet fed ApoE-/- mice. To further investigate the role of epsins in atherosclerosis, we created endothelial cell-specific epsin deficient ApoE-/- mice (ApoE-/-/EC-DKO) by crossing Epn1fl/fl, Epn2-/- mice with constitutive VEcad Cre mice then backcrossed and bred them to an ApoE-/- background. When fed western diet, and compared to ApoE-/- /WT mice, we found that ApoE-/-/EC-DKO mice were resistant to diet-induced atherosclerosis. Development of the atheroma is dependent, in part, on endothelial activation, i.e. upregulated expression of adhesion molecules, selectins, chemokines and cytokines, which facilitate the recruitment of immune and inflammatory cells. TNFR-NF-?B signaling and the subsequent downstream transcriptional activation, is a critical cascade necessary for endothelial activation. Therefore, we describe a research strategy to test the central hypothesis that epsins play a critical role in atherosclerosis by promoting TNFR-NF-?B signaling resulting in inflammation and subsequent recruitment of immune/inflammatory cells to the developing atheroma. In support, we report that epsin deficiency impaired TNF-induced TNFR-NF-?B signaling in aortic endothelial cells in vitro. Impaired activation of epsin deficient endothelial cells corresponded with decreased macrophage rolling/adhesion in vitro, which is a critical step in macrophage recruitment, infiltration and foam cell formation. Furthermore, we have preliminary data implicating a novel regulatory mechanism for epsins in TNFR signaling complex assembly, independent of its conventional role as an endocytic adaptor. Therefore, we also propose a strategy to characterize the molecular mechanism by which epsins promote TNFR-NF-?B signaling. Specifically, our data suggests epsins play a novel and critical role in recruiting and/or stabilizing TNFR signaling complex formation. In summary, the information gained from this proposal will provide a novel regulator, and potential new therapeutic target, for the development and progression of atherosclerosis. In addition, it will result in the identificatin and characterization of a novel function for epsins, independent of their role as endocytic adaptors.